Polymer with superior polar retention for sample pretreatment

ABSTRACT

A polymeric sorbent that can be employed in the extraction and purification of polar and nonpolar molecules from a complex media (e.g. pharmaceuticals from biological matrices) by solid phase extraction (SPE). The sorbent exhibits a strong capacity for the retention of polar molecules and can facilitate the recovery of compounds possessing a range of polarities while furnishing clean extracts showing low ion suppression. The polymer is wettable and remains wetted over long periods of time.

CROSS REFERENCE TO RELATED APPLICATIONS

The present patent application is based on and claims priority to U.S.Provisional Application Ser. No. 60/385,604, entitled “A POLYMER WITHSUPERIOR POLAR RETENTION FOR SAMPLE PRETREATMENT”, which was filed Jun.3, 2002 and is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to a functionalized polymeric sorbent, useful forisolating an analyte of interest from an aqueous or biological matrix,through a solid phase extraction (SPE)-based sample pretreatment. Thepolymeric sorbent strongly retains moderately to highly polar molecules,in addition to hydrophobic compounds. The present invention also relatesto a method for extraction/clean-up with this polymeric material, aswell as the preparation and use of the polymeric material. The polymericsorbent can be employed in separations and purifications in variousfields, for example the pharmaceutical, diagnostic, environmental,toxicological, clinical, nutritional and agrochemical fields.

Abbreviations ESI electrospray ionization FTIR Fourier-transforminfrared GC gas chromatography HPLC high performance liquidchromatography LLE liquid-liquid extraction LC liquid chromatography MSmass spectrometry NMR nuclear magnetic resonance PS-DVB poly(styrenedivinylbenzene) SPE solid phase extraction

BACKGROUND OF THE INVENTION

Sample preparation is a critical step in the analysis of complexmatrices for trace components, particularly in the area of lifesciences. Solid phase extraction (SPE) techniques can be valuable to ananalyst solving problems relating to sample concentration, sampleclean-up and analyte isolation. SPE is recognized as a desirablealternative to liquid-liquid extraction (LLE) because SPE minimizes oreliminates altogether the use of organic solvents, which are regulatedas priority pollutants. Further, LLE can lead to emulsion formation andif particulates are present in a sample, adsorption of analyte ontothese structures can result in low recoveries. Compared with LLE, SPEcan offer a more complete extraction of analytes, a more efficientseparation of interferences from analytes, easier collection of totalanalyte fraction and removal of particulates and can be more easilyautomated. Solid phase extraction is presently extensively applied inseparations performed in widely differing fields, including, but notlimited to environmental pollution, agrochemicals, discovery and/ordevelopment of pharmaceuticals, analytical toxicology, the developmentof nutritional products, drinking water purity assessment andbiotechnology. Several individual monographs, journal review articlesand research publications on the theory and practice of SPE technologyhave been published (see, e.g., Thurman & Mills, (1998) Solid PhaseExtraction, Wiley, New York, N.Y.; Simpson (Ed.), (2000) Solid PhaseExtraction, Marcel Dekker, New York, N.Y.; J Chromatog. A. (2000) 885:entire issue; Snyder, Kirkland & Glajch, Practical HPLC MethodDevelopment, Chapter 4, pp 100-173, Wiley, New York, N.Y., 1997).

Solid phase extraction protocols followed by academic, industrial andgovernment laboratories typically employ syringe-barrel cartridges,which can include cartridges designed for syringe use, as well as disksand disk cartridges (see, e.g., Thurman & Snavely, (2000) Trend Anal.Chem. 19:18-26), thin packed bed syringe-barrel cartridges, solid phasemicroextraction fibers (for both gas chromatographic (GC) and highperformance liquid chromatography (HPLC) applications), 96-well plates,SPE pipette tips, and robot-compatible large reservoirs. The syringebarrel device format is the most commonly employed format, followed bythe disk format. The disk format facilitates the use of higher flowrates, due to their large cross-sectional areas and shorter bed depths,and utilize very small elution solvent volumes. For drug screening andclinical trial applications, both of which require high samplethroughput and utilize liquid chromatography/mass spectrometry/massspectrometry (LC/MS/MS) as the primary analytical tool, the multi-wellplate format (e.g. 96-well plates, 384-well plates and 1536-well plates)has gained popularity.

Silica and related bonded phases constituted the dominant SPE sorbentsuntil about 1996, as evidenced by the extensive applicationbibliographies prepared by several SPE material manufacturers (e.g.,Varian Sample Preparation Products, Harbor City, Calif., 1995; BakerbondSPE Bibliography, JTBaker, Inc, Philipsburg, N.J., 1995; McDonald &Bouvier, (Eds.), Solid Phase Extraction Applications Guide andBibliography: A Resource for Sample Preparation Methods Development,Waters Corp., Milford, Mass. 6^(th) ed., 1995). During the last fewyears, however, many polymeric sorbents have been introduced for samplepretreatment applications (see, e.g., U.S Pat. No. 5,618,438; U.S. Pat.No. 5,882,521; and U.S. Pat. No. 6,106,721; Fritz & Macka, (2000) J.Chromatog. A 902:137-166). Some of these polymeric sorbents are based ona styrene divinylbenzene or methacrylate polymeric backbone. Advantagesof polymeric SPE sorbents over their silica-based counterparts includetheir stability to pH extremes and their higher surface area, which canfacilitate greater capacity and retention than observed for silica-basedmaterials. In addition, silica-based materials comprise silanol groups.These groups can complicate analyte retention, due to the influence ofthe pH and ionic strength of the sample matrix on the silanol groups.

One limitation of commercially available reversed-phase silica sorbents,as well as the first generation of styrene-divinylbenzene polymers, isthe need for conditioning them with a wetting solvent and the additionalrequirement that they remain wetted prior to sample loading. The adventof second generation polymeric sorbents comprising polar functionalgroups such as sulfonic/carboxylic acid, hydroxymethyl, keto, nitro andheterocyclic amide moieties ameliorates these requirements due to thecapacity of these polar groups to adsorb and retain water on theirsurface.

These reversed-phase silica and second generation polymeric materialsare not, however, without problems. A major shortcoming ofreversed-phase silicas and second generation polymers is the inabilityof these materials to retain polar compounds, such as some drugmetabolites and pharmaceuticals. Many of these SPE materials exhibitunacceptable breakthrough for polar molecules during the loading and/orwashing steps, resulting in poor analyte recoveries. This phenomenonplaces severe limitations on the applicability of SPE protocols foranalyte extraction and sample clean-up when the sample comprises amixture of an analyte, which can be hydrophobic, and its metabolites ordegradation products, which tend to be very polar. Moreover, thepharmaceutical industry is designing more products with significantpolar characteristics. The inadequate retention of such drugs on apolymeric sorbent during sample pretreatment can lead to seriousproblems.

Another limitation of prior art polymeric sorbents is in the area of ionsuppression. Several publications highlight an ion suppression effectobserved during LC/MS/MS analysis of drugs in biological matrices (see,e.g., Bonfiglio et al., (1999) Rapid Commun. Mass Sp. 13: 1175-1185;King et al., (2000) J Am. Soc. Mass Spectr. 11: 942-950). Thesepublications attribute the observed ion suppression to the presence ofmatrix constituents left behind on an SPE sorbent during sample loadingand washing steps. These constituents can then contaminate desiredextracts during analyte elution. During LC/MS, polar drugs elute fromthe LC column either with these matrix constituents or closely afterelution of the matrix constituents. These polar drugs can be severelyaffected by ion suppression, rendering their quantitation unreliable.Thus, another problem associated with prior art sorbents is the presenceof unacceptable levels of ion suppression.

Yet another problem associated with prior art SPE materials is thelimitation on the amount of organic component that can be employed towash (or elute) an analyte of interest after a sample comprising theanalyte is applied to a prior art polymeric sorbent. Procedures foremploying prior art SPE materials typically recommend the use of aqueoussolvents and buffers containing a low percentage of an organic component(<5%) for washing the SPE material after a sample has been loaded ontothe material. These procedures recommend a low percentage of organiccomponent because if the organic content is increased too much, this canlead to the almost complete removal of the more polar constituents ofthe sample, including an analyte of interest. This is due, in part, tothe inability of prior art sorbents to retain moderately to highly polarcompounds. A few commercial polymeric sorbents, such as those comprisingsulfonic acid moieties, are known to enhance polar retention throughionic mechanisms. SPE protocols using these sorbents are tedious,however, and such elutions are typically carried out with solvents thatare incompatible with mass spectrometric detectors.

Thus, there is a need for a polymeric sorbent that strongly retainsmoderately to highly polar analytes, particularly when the analytes arepresent in a complex matrix (e.g. a biological, environmental orpharmaceutical sample). There is also a need for a polymeric sorbentthat can be treated with solvents comprising a high percentage of anorganic component, such that after sample loading, the sorbent can bewashed thoroughly with an aqueous-organic binary solvent containing areasonably high percentage of organic. Such a wash would furnish a cleanextract by removing unwanted matrix components, which can interfere withmass spectrometric detection and cause ion suppression. An SPE protocolemploying this sorbent would preferably comprise a simple procedure forelution of the desired analyte, such that the eluting solvent iscompatible with mass spectrometric mode of detection and if necessary,be adapted to be injected directly into an LC/MS/MS system. Further,such a sorbent would preferably be easily solvated with an aqueoussolvent (e.g. water or buffer), remain solvated for a long period oftime and would display comparable SPE behavior under wet or dryconditions. Such an SPE procedure/format would preferably be compatiblewith the high throughput screening of large volume of samples commonlyemployed in the pharmaceutical industry. These and other problems aresolved by the compositions and methods of present invention.

SUMMARY OF THE INVENTION

In one aspect of the present invention, a polymeric sorbent isdisclosed. In one embodiment, the polymeric sorbent comprises: (a) apolymeric backbone adapted to facilitate one or more interactionsselected from the group consisting of a dipolar interaction and ahydrophobic interaction; and (b) an amide functionality associated withthe polymeric backbone and adapted to undergo one or more interactionsselected from the group consisting of proton accepting, proton donatingand dipolar interactions.

In this and other embodiments of the present invention, a polymericbackbone of a sorbent of the present invention can comprise, forexample, poly(styrene divinylbenzene), copolymers of styrene, copolymersof divinylbenzene, functionalized styrenes, functionalized heterocyclesand combinations thereof. An amide functionality of a sorbent of thepresent invention can comprise, for example, acetamide, N-alkylamides,N-aryl amides and N-heteryl amides.

In this and other embodiments of the present invention, a polymericsorbent of the present invention can comprises between about 3.5% andabout 5.0% nitrogen by mass percent and can comprise particles having acharacteristic dimension (e.g. diameter) of between about 20 and about120 microns. In another aspect, a polymeric sorbent of the presentinvention can remain solvated after contact with a solvent for longerthan about one hour and can adsorb strongly polar, moderately polar andnonpolar molecules. In another aspect, a polymeric sorbent of thepresent invention, can be associated with a support, such as acartridge, a polymeric fiber membrane, a glass fiber membrane and amulti-well plate.

In another aspect of the present invention, a method of preparing apolymeric sorbent functionalized with an amide functionality isdisclosed. In one embodiment, the method comprises: (a) nitrating apolymeric backbone to form a nitrated polymeric backbone; (b) reducingthe nitrated polymeric backbone to form an aminated polymeric backbone;and (c) contacting the aminated polymeric backbone with one of an acid,an acid chloride and an acid anhydride.

In one embodiment, the nitrating comprises: (a) suspending a polymericbackbone in a first solution comprising nitric acid; and (b) adding asecond solution comprising a reagent adapted to generate a nitronium ionto the first solution. In one embodiment, the reducing comprises: (a)suspending a nitrated polymeric backbone in a first solution comprisinga first acid; and (b) contacting the nitrated polymeric backbone with asecond solution comprising a metal catalyst and a second acid.Continuing, in one embodiment, the contacting comprises: (a) suspendinga reduced polymeric backbone in a first solution comprising a base toform a basic reaction solution; and (b) adding one of an acid, an acidchloride and an anhydride to the basic reaction solution. The disclosedmethod is not limited to the recited steps and can further comprise, forexample, the steps of (a) recovering the polymeric sorbent byfiltration; (b) washing the polymeric sorbent one or more times with asolution comprising an acid; (c) washing the polymeric sorbent one ormore times with an aqueous solution; and (d) washing the polymericsorbent one or more times with an organic solvent.

In yet another aspect of the present invention, a method of isolating ananalyte from a sample is disclosed. In one embodiment, the methodcomprises: (a) conditioning the sorbent by washing the sorbent with anorganic solvent followed by water; (b) contacting a sample comprising ananalyte disposed in an aqueous medium with a polymeric sorbentcomprising (i) a polymeric backbone adapted to form at least one of adipolar interaction and a hydrophobic interaction; and (ii) an amidefunctionality associated with the backbone and adapted to undergo protonaccepting and proton donating interactions; to form a sorbent-samplecomplex; (c) washing the sorbent-sample complex with water followed byan organic solvent; and (d) eluting an analyte from the sorbent-samplecomplex with an eluting solvent, whereby an analyte is isolated from asample. A sample can be, for example, a biological matrix comprising ananalyte, an environmental sample, an aqueous pharmaceutical sample or anaqueous nutraceutical sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram generally depicting a synthetic protocolby which a polymeric sorbent of the present invention can besynthesized.

FIG. 2 is an FTIR spectrum of a polymeric sorbent of the presentinvention; the sorbent comprises an acetamide functionality.

FIG. 3A is a solid-state ¹³C NMR spectrum of a polymeric sorbent of thepresent invention; the sorbent comprises an acetamide functionality.

FIG. 3B is solid-state ¹³C NMR of the a [poly(styrene divinylbenzene)]polymer.

FIG. 4 is a plot depicting the minimal breakthrough of moderately andstrongly polar compounds associated with a polymeric sorbent of thepresent invention during the wash step of an SPE protocol.

FIG. 5A is a plot depicting the typical retention profile of polar andhydrophobic compounds by prior art silica and polymer-based SPEsorbents.

FIG. 5B is a plot depicting the preferred retention of polar andhydrophobic compounds by a sorbent during a wash step of an SPEprotocol.

FIG. 6 is a cartoon depicting some polar and hydrophobic interactionsthat a polymeric sorbent of the present invention can undergo withvarious analyte functionalities.

FIG. 7 is a bar graph depicting the effect of post-conditioning dryingtime on the recovery of seven analytes applied to a polymeric sorbent ofthe present invention; the analytes were recovered immediately afterconditioning the sorbent (black bars) and after a 1 hour drying periodfollowing conditioning (white bars).

FIG. 8 is a bar graph depicting the recovery of seven analytes from aspiked canine plasma sample which was treated by following a protocolemploying a polymeric sorbent of the present invention, as detected byLC/MS/MS. Black bars represent a 400 □l elution from a polymeric sorbentof the present invention; dark gray bars represent a 1 ml elution from apolymeric sorbent of the present invention; light gray bars representelution from the OASIS® sorbent (Waters Corporation, Milford, Mass.).

FIG. 9 is a cleanliness check plot in the mass range 500-2200 comparingthe purity of a bovine plasma extract isolated by employing a polymericsorbent of the present invention with the purity of the same sampleisolated by employing a commercially available prior art polymericsorbent in the mass range 500-2200.

FIG. 10 is a cleanliness indicator plot in the mass range 400-600comparing the purity of a bovine plasma extract isolated by employing apolymeric sorbent of the present invention with the purity of the samesample isolated by employing a commercially available prior artpolymeric sorbent.

FIG. 11 is a time versus intensity (represented as counts) plotdepicting the ion suppression associated with a bovine plasma extractfrom a polymeric sorbent of the present invention and the ionsuppression associated with a commercially available prior art polymericsorbent.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this application, including the claims.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of ±20% or ±10%, more preferably ±5%, evenmore preferably ±1%, and still more preferably ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, the term “adsorb”, and grammatical derivatives thereof,means a surface phenomena wherein an analyte becomes reversiblyassociated with the surface of a polymeric sorbent by physicallyinteracting with the surface molecules. The association can be, forexample, via any non-covalent mechanism (e.g. van der Waal's forces,such as dipole-dipole interactions, dipole-induced dipole or dispersiveforces, via hydrophobic interactions or hydrogen donor or acceptorinteractions).

As used herein, the term “acid chloride” means a chemical entitycomprising a variable organic group (R1, which can comprise hydrogen, oran alkyl, aryl or heterocyclic moiety), a carbonyl and a chlorine atom,and is represented by the chemical structure

As used herein, the terms “amide,” “amide group” and “amidefunctionality” are used interchangeably and mean a chemical entitycomprising a carbonyl, a variable organic group (R1) joined to thecarbonyl and a group comprising a nitrogen atom and at least twoindependently variable organic groups (R2 and R3, which can comprisehydrogen or an alkyl, aryl or heterocyclic moiety), and can berepresented by the chemical structure

In the compositions and methods of the present invention, for example, apreferred amide is acetamide, represented by the chemical structure

wherein R1 represents a polymeric backbone. Broadly, then, the term“amide functionality” means any chemical entity comprising an amidegroup.

As used herein, the term “analyte” means any molecule of interest. Ananalyte can comprise any polarity, although in the context of thepresent invention, moderately polar to highly polar molecules are ofparticular interest. An analyte can be disposed in a sample, and canform a component thereof. For example, a candidate therapeutic compoundor metabolic byproducts thereof, can be an analyte, and the analyte canbe disposed in, for example, a blood plasma sample, saliva, urine,drinking water, and water known or suspected to be polluted. Summarily,an analyte can comprise any molecule of interest.

As used herein, the term “anhydride” means a chemical entity comprisingtwo carbonyls and two variable organic groups (R1 and R2, which cancomprise an alkyl, aryl or heterocyclic group), which can independentlybe the same or different, and can be represented by the chemicalstructure

As used herein, the term “associated” means a joining of two or morechemical entities. An association can be via a covalent or vianon-covalent bond (e.g., hydrophobic interaction, hydrogen bonding,ionic interactions, van der Waals' forces and dipole-dipoleinteractions).

As used herein, the terms “support” and “supporting format” are usedinterchangeably and mean a porous or non-porous water insolublematerial. A support or a supporting format can have any one of a numberof configurations or shapes, such as strip, plate, disk, rod, particle,including bead, and the like. A support or supporting format can behydrophobic, hydrophilic or capable of being rendered hydrophilic, andcan comprise inorganic powders such as silica, zirconia, and alumina;natural polymeric materials, synthetic or modified naturally occurringpolymers, such as nitrocellulose, cellulose acetate, poly (vinylchloride), polyacrylamide, polyacrylate, polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), nylon, poly(vinyl butyrate), polytetrafluoroethylene,etc.; either used by themselves or in conjunction with other materials;glass available as Bioglass, ceramics, metals, and the like (see, e.g.,Buchmeiser, (2001) J. Chromatog. A 918:233-266). Natural or syntheticassemblies such as liposomes, phospholipid vesicles, and cells can alsobe employed.

As used herein, the term “strongly polar” means a molecule that, basedon the octanol-water partition coefficient log P, has a log P value of−1.0 to +0.5.

As used herein, the term “moderately polar” means a molecule that, basedon the octanol-water partition coefficient log P, has a log P value of0.5 to 1.5.

As used herein, the term “nonpolar” means a molecule that based on theoctanol-water partition coefficient log P, has a log P value greaterthan or equal to 2.0.

II. General Considerations

An aspect of the present invention is the development of a polymericsorbent that strongly retains moderate to highly polar molecules (e.g.pharmaceuticals, such as sulfa drugs, atenolol, ranitidine andpseudoephedrine). The retention profile of this polymer allows ananalyst to load a sample onto a sorbent and subsequently subject theloaded sorbent to a very thorough wash with a binary solvent comprisingan aqueous component and an organic component present, which can removemany unwanted components of the sample. Preferably the organic component(e.g. acetonitrile or methanol) is present in a high percentage(e.g. >about 10-30% organic), thereby facilitating the elimination ofmatrix constituents from biological or environmental samples completelyand the ability to obtain purer extracts.

In the present disclosure, when referring to the polarity of a molecule(e.g. a “strongly polar” molecule, a “moderately polar” molecule, a“nonpolar” molecule, etc.), polarity is described based on the standardoctanol-water partition coefficient, P. This coefficient is sometimesexpressed as log P. The octanol-water partition coefficient is a measureof polarity commonly known and used by those of ordinary skill in theart. Standard methods of determining octanol-water partitioncoefficients are known (see, e.g., Sangster, (1997) Octanol-WaterPartition Coefficients: Fundamentals and Physical Chemistry, Wiley,Hoboken N.J.) and many P values have been tabulated (see, e.g.,Sangster, (1989) J. Phys. Chem. Ref. Data 18(3): 1111-1230; Howard &Meylan, (eds.) 1997 Handbook of Physical Properties of OrganicChemicals, Lewis, Boca Raton, Fla.). Specific definitions for somepolarity descriptors are provided herein.

The isolation process can also minimize or eliminate any ion suppressiondue to interference of matrix constituents with the ionization processof analytes under investigation. The elimination of ion suppression isof benefit to operations involving electrospray ionization (ESI), forexample, which is commonly employed in LC/MS/MS analyses. Additionally,since sample solutions treated by SPE protocols are predominantlyaqueous in nature, solvation of the sorbent surface (commonly calledwettability or hydration when water is employed) can be desirable, andcan play a role in the retention of polar analytes. The polymericsorbents of the present invention are readily solvated and can remainsolvated for long periods of time (greater than about one hour).

One commercially available prior art polymeric sorbent that isextensively used in the pharmaceutical industry for sample cleanup is acopolymer comprising divinylbenzene and N-vinyl pyrrolidone. It has beenstated that the introduction of the pyrrolidone component into theoverall divinylbenzene-N-vinylpyrrolidone copolymeric structure providesan interactive surface for polar drugs (Bouvier et al., (1998), LC.GC(Supplement), May 1998, pp S53-S58). A number of literature publicationsindicate, however, that when this prior art copolymer is employed in anSPE protocol, moderate to strongly polar analytes are not adequatelyretained and that complex pH-controlled extraction procedures arerequired in order to enhance the retention of moderate to strongly polaranalytes on this sorbent (see, e.g., Cheng et al., (1999) J. Chromatogr.B 729: 19-31; Georga et al., (2001) J. Chromatogr. B 759: 209-218).Additionally, SPE extracts from this polymeric sorbent were found tocontain impurities, presumably due to strong adsorption of matrixconstituents of biological samples applied to the sorbent (see, e.g.,Zheng et al., (2002) J. Pharm. Biomed. Anal. 28: 279-285.)

Another prior art polymeric sorbent that has been developed comprisesthe same divinylbenzene N-vinylpyrrolidone basic skeleton but alsocomprises a sulfonic acid moiety. This sorbent retains basic analytesvia an ionic mechanism but, like other prior art polymeric sorbents,pH-controlled solvent systems are required to load, wash and elute basicdrugs from this prior art sorbent (see, e.g., Kollroser & Schober,(2002) J. Chromatogr. B 766: 219-226). The purity of extracts of polardrugs from serum, obtained from this sulfonated polymer, was still foundto be unacceptable, in spite of the fact that the sorbent can besubjected to strong solvent wash after the loading step (Muller et al.,(2002) J. Chromatogr. B 773: 47-52). Further, when acidic analytes frombiological matrices are to be purified, this sulfonated polymer isinefficient and an anion exchange sorbent is preferably employed.

The retention of analytes comprising various degrees of polarity andhydrophobicity on both silica and polymer-based SPE sorbents (excludingion-exchange resins) is presented in FIG. 5A. This figure was generatedby pooling SPE data on a wide range of compounds of different polaritiesdocumented in literature, (Hennion, (1999) J. Chromatogr. A 856: 3-54;Casas et al., (1992) Chromatographia 34: 79-82; Pichon et al., (1998) JChromatogr. A 795: 83-92; Hennion et al., (1998) J. Chromatogr. A 823:147-161) as well as from unpublished data recorded in the inventors'laboratories. FIG. 5A demonstrates that analytes of moderate to strongpolarity show considerable breakthrough on both silica-based reversedphases and polymeric sorbents, indicating that these analytes are notretained well on these sorbents. By way of comparison, FIG. 5B depicts apreferred retention profile for polar and hydrophobic molecules. Thisfigure depicts a hypothetical profile indicating how an ideal SPEsorbent is predicted to perform, although no state-of-the-art sorbentexhibits such an ideal behavior. In this retention profile, after asample loading step, the sorbent is amenable to washing with an aqueousbinary solvent comprising an aqueous component and an organic componentcomprising about 20% or more organic (e.g. acetonitrile or methanol).

Thus, commercially available prior art polymeric sorbents are unable toeffectively retain moderately to highly polar analytes. Further, thesesorbents are not amenable to washing with a binary solvent comprising ahigh percentage of an organic component, which can limit the purity ofan eluted analyte. As described hereinbelow, however, the polymericsorbents of the present invention, on the other hand, meet thesecriteria and can be employed to isolate moderate and highly polaranalytes.

III. Theoretical Considerations for Designing a Polymeric Sorbent of thePresent Invention

When designing a sorbent with enhance polar retention, the solvationparameter model equation (1) can be considered.Log SP=c+mV _(x) +rR ₂ +sπ₂ ^(H) +aΣα₂ ^(H) +bΣβ₂ ^(H)  (1)wherein SP is a solute property, such as capacity factory (k′) orbreakthrough volume or elution volume; the solute descriptors are V_(x),which represents molecular volume; π₂ ^(H), which representsdipolarity/dipolarizability; and Σα₂ ^(H) and Σβ₂ ^(H), which representa solute's effective hydrogen-bond acidity and hydrogen-bond basicity,respectively. The other parameters in the equation represent the system,which is a combination of the sorbent and the solvent. The m term is thecapacity of the sorbent to form a cavity adapted to accommodate thesolute. The system constant r represents the difference in the capacityof the sorbent and sample solution to interact with n or π electrons ofthe solute. The system constant s represents the difference in thecapacity of the sorbent and sample solution to take part indipole-dipole and dipole-induced dipole interactions. The constantdenotes the difference in hydrogen-bond basicity of the sorbent andsolution and the b constant denotes the difference in hydrogen-bondacidity of the sorbent and solution.

In equation (1), c is a constant, which is characteristic of the system.The two terms mV_(x) and rR₂ represent the steric fit and hydrophobicinteractions, respectively, between the solute and the sorbent. Theother parameters, namely sπ₂ ^(H), aΣα₂ ^(H) and bΣβ₂ ^(H), representpolar interactions resulting from dipole-dipole, solute acidity-sorbentbasicity and solute basicity-sorbent acidity interactions, respectively.

To enable a sorbent to interact with and retain an analyte, a sum ofthese interactions can be considered. For polar analytes in particular,the abovementioned polar interactions of the sorbent surface with theseanalytes can be of great significance with respect to analyte retention.For example, polar analytes comprise acidic and/or basic functionalgroups. In some cases polar analytes comprise a neutral, but stronglypolar, moiety such as a glucuronide, an amide or a sulfonamide. In orderto retain analytes with such functionalities, the sorbent preferablycomprises hydrogen bond donor (acidic) or hydrogen bond acceptor (basic)sites in its structure. Furthermore, the strength of hydrogen bondsarising from solute-sorbent interactions are preferably higher thansimilar bonds a solute or sorbent can form with water or methanol, whichcan facilitate retention of polar analytes during a wash step of anoverall sample purification and/or extraction protocol. In addition, itis preferable that a functional group meeting these goals is located onthe polymeric backbone of a sorbent. It is also preferable that thepolymeric backbone itself can also simultaneously undergo dipole-dipoleinteractions with the □ electrons of an unsaturated group or an aromaticsystem present on a polar analyte.

A survey of the applicable literature indicates that of the variousfunctional classes of organic compounds, an amide functionality carryingat least one hydrogen atom on the nitrogen meets all the three criteriaoutlined above, namely dipole-dipole interactions, hydrogen bondbasicity and hydrogen bond acidity. By way of specific example,formamide shows a π value of 0.46, a Σα value of 0.33 and a Σβ value of0.2, while the corresponding values for N,N dimethylformamide are 0.56,0.00 and 0.44, respectively. For N-methyl pyrrolidone, the correspondingfigures are 0.57, 0.00 and 0.43, respectively. The log P value ofacetanilide is 1.16, while those of toluene, methyl benzoate andacetophenone are 2.74, 2.18 and 1.66, respectively. These values showthat the amide functionality is the most polar amongst different classesof substituted benzenes. The hydrogen bond forming capabilities of theamide functionality is also evident from nucleic acid and proteinchemistry. Thus, a polymeric sorbent adapted to retain a moderately tohighly polar analyte, such as the polymeric sorbents of the presentinvention, preferably comprise an amide functionality.

IV. A Polymeric Sorbent of the Present Invention

A polymeric sorbent of the present invention broadly comprises apolymeric backbone adapted to facilitate one or more interactionsselected from the group consisting of dipolar interactions and ahydrophobic interactions; and an amide functionality associated with thepolymeric backbone and adapted to undergo one or more interactionsselected from the group consisting of proton accepting, proton donatingand dipolar interactions.

Any polymer adapted to form at least one of a dipolar interaction and ahydrophobic interaction can be employed as a polymeric backbone in thepresent invention. A polymeric backbone can comprise, for example,poly(styrene divinylbenzene), copolymers of styrene or divinylbenzenewith functionalized styrenes or heterocycles carrying substituents suchas halo, alkoxy, ester or nitro; or copolymers such as (but notrestricted to) polystyrene-polyacrylamide and polystyrene-polyacrylates.Thus, a representative, but non-limiting, list of polymers that can beemployed as a polymeric backbone in a sorbent of the present inventionincludes, but is not limited to, poly(styrene divinylbenzene),copolymers comprising styrene or divinylbenzene and methylmethacrylate,halogenated or nitrated or aminated or hydroxylated styrenes,functionalized isocyanurates, urethanes, acrylamides or acrylonitrilesand functionalized heterocyclic systems, such as vinyl/allyl pyridines.In one embodiment, a polymeric backbone comprises poly(styrenedivinylbenzene), a ¹³C NMR spectrum of which is presented in FIG. 3B. Itis preferable that the polymeric backbone comprises spherical particleshaving a characteristic dimension (e.g. a diameter) of between about 20and about 120 microns in diameter. Although non-spherical or irregularparticulate polymers can be employed in the present invention, it ispreferable that polymers comprise spherical particles, which arecommercially available and can be readily employed in the preparation ofa polymeric sorbent of the present invention. Spherical particulatepolymers can readily form slurries, exhibit better flow characteristics,can be packed more uniformly and possess greater mechanical stability,which can be desirable in SPE protocols. When a polymeric backbonecomprises particles, the particles can be porous and the particles cancomprise a pore size of, for example, between about 50 to about 150angstroms or, for example, between about 50 to about 70 angstroms.

A polymeric sorbent of the present invention also comprises an amidefunctionality associated with the polymeric backbone and adapted toundergo one or more interactions selected from the group consisting ofproton accepting, proton donating and dipolar interactions, for examplewith the functionalities of an analyte. Representative amidefunctionalities include acetamide, N-alkylamides, N-aryl-amides andN-heteryl amides. Some of the interactions that can occur between anamide functionality and the functional groups of some representativefunctionalities that are found on an analyte are illustrated in FIG. 6.These interactions can contribute to the retention of different classesof analytes.

An amide functionality can be associated with the polymeric backbone,via a covalent bond, for example, and can be associated at one or moreidentical positions on the length of a polymeric backbone. Although theconfiguration of an amide functionality of a polymeric sorbent of thepresent invention can vary, the nitrogen atom of an amide functionalityis preferably associated with a polymeric backbone at one point, ahydrogen atom at another point and a variable organic group at anotherpoint as depicted:

The variable organic group comprises at least one carbonyl, which formsan element of an amide functionality. Although any organic group canform a component of an amide in a sorbent of the present invention, arepresentative, but non-limiting, list of variable organic groups (whichcan be associated with a carbonyl) includes methyl, higher alkyl orcycloalkyl, phenyl/alkylphenyl/functionalized phenyl or napthyl orhigher polyaromatic ring systems and similarly substituted heterocyclicgroups.

A poly(styrene divinylbenzene) polymeric backbone substituted with anacetamide functionality comprises one polymeric sorbent of the presentinvention. The inclusion of an acetamide functionality in a polymericsorbent of the present invention affords the sorbent a balance betweenthe retention of polar and hydrophobic drugs under solid phaseextraction conditions when compared with other amide derivatives.

In one embodiment, a percentage of nitrogen in a polymeric sorbent ofthe present invention is between about 3.5% and about 5.0% by masspercent for the retention of both hydrophilic and hydrophobic analytes.In another embodiment, the nitrogen content of a sorbent is betweenabout 4.0% and about 4.5% by mass percent.

In one embodiment of a polymeric sorbent of the present invention, thesorbent can be associated with a support. Some examples of supportsinclude syringe barrel cartridges and multi-well plates (see, e.g., U.S.Pat. No. 6,200,533), although disks, membranes (see, e.g., U.S. Pat. No.5,738,790), tubes (see, e.g., U.S. Pat. No. 5,137,626) and othersupports can also be employed.

A polymeric backbone (and subsequently a polymeric sorbent of thepresent invention) can, but need not, comprise particles having acharacteristic dimension (i.e., diameter) of between about 20 and about120 microns. In other examples, a polymeric backbone can comprise a poresize of between about 50 to about 150 angstroms, or between about 50 toabout 70 angstroms.

V. Preparation of a Polymeric Sorbent of the Present Invention

In one embodiment of the present invention, poly(styrene divinylbenzene)(PS-DVB) was functionalized by the introduction of an amidefunctionality by a three step synthetic sequence, as shown in FIG. 1 andas described in Laboratory Example 1. The starting polymer, poly(styrenedivinylbenzene), is commercially available from several manufacturers asuniform spherical particles (e.g., Polymer Laboratories of Amherst,Mass.; Tosoh Haas of Stuttgart, Germany; and Shodex of Tokyo, Japan).

In the first step of a representative synthetic sequence, PS-DVB can benitrated with a mixture of nitric and sulfuric acids under optimalconditions to yield a nitrated poly(styrene divinylbenzene). Althoughthe introduction of a nitro moiety into an aromatic nucleus ofpoly(styrene divinylbenzene) is known in literature, (see, e.g.,Philippides et al., (1993) Polymer 34: 3509-3513) the proceduresreported are tedious and the conditions employed are drastic. Forexample, in one prior art method, dimethylformamide was used as asolvent to suspend the polymer and a mixture of fuming nitric acid andsulfuric acid was used and the reaction was initially carried out at2-5° C. for 3 hours, followed by heating at 60° C. for 6 hours(Philippides et al., (1993) Polymer 34: 3509-3513).

In one aspect of the present invention, on the other hand, a simpler andless time-consuming protocol is disclosed. In an example of this aspectof the present invention, a polymeric backbone (poly(styrenedivinylbenzene), for example) is suspended in nitric acid and a strongacid (e.g. sulfuric acid) is added over about 1 to about 1.5 hours. Themixture is stirred at room temperature for about 3 hours, therebyforming a nitrated polymeric backbone. Additional discussion of thisaspect of the present invention is presented in Laboratory Example 1.Representative amounts of nitric and sulfuric acids used in thenitration reaction are about 25 to about 35 and about 15 to about 20moles, respectively. During the course of the nitration, stirring can bemaintained, and when stirring is maintained a mechanical stirreroperating at a speed of about 100 to about 150 rpm can be employed.Higher stirring speeds might cause breakage of the polymer particles andintroduced a high percentage of fines into the product.

In another step of the synthesis of a functionalized poly(styrenedivinylbenzene) sorbent of the present invention, the nitrated polymercan be reduced to an amino group by reduction with a catalyst (e.g.stannous chloride) and an acid (e.g. hydrochloric acid), thereby formingan aminated polymeric backbone. The reduction can be performed at roomtemperature and can be accompanied by stirring. Additional discussion ofthis aspect of the present invention is presented in LaboratoryExample 1. Again, the stirring speed can be regulated to preventbreakage of the polymer spheres.

In another step of the synthesis of a functionalized poly(styrenedivinylbenzene) sorbent of the present invention, an aminated polymericbackbone, such as poly(styrene divinylbenzene), is derivatized(acylated, for example) with an acid, an acid chloride or an acidanhydride to yield a desired amide functionalized polymer.

The synthesis of a sorbent of the present invention can optionallycomprise additional steps. For example, following the step of contactingan aminated polymeric backbone with an acid, acid chloride or acidanhydride, the resultant sorbent can be recovered by filtration.Filtration through a suitable structure, such as a membrane, can removethe sorbent from solution, making it easy to subsequently treat. Thesorbent can then be subjected to additional washes to remove undesiredcomponents. Such washes can comprise washing one or more times with anacid, followed washing one or more times with an aqueous solution andwashing one or more times with an organic solvent. Cumulatively, thesewashings can not only remove undesired components that might beassociated with the sorbent, but the can also place the sorbent inconditions for either temporary or long term storage until use.

In another aspect of the present invention, several structural analogueswere synthesized and screened for their retention characteristics forpolar drugs. For example, an aminated poly(styrene divinylbenzene)sorbent of the present invention can be treated with one or more of (a)4-nitrobenzoyl chloride to yield a 4-nitrobenzoyl amide derivative ofthe polymer; (b) 4-acetamido benzoylchloride to furnish4-acetamidobenzoylamide derivative of the polymer; (c) 2-furoyl chlorideto provide 2-furoyl amide substituted polymer; and (d) acetic anhydride,in order to generate an acetylamido functionalized poly(styrenedivinylbenzene) sorbent of the present invention.

In the preparation of these exemplary compounds, acid chlorides can beemployed to introduce the variable organic moiety. In anotherembodiment, corresponding anhydrides in the presence of a base catalystcan also be employed. In yet another embodiment, a carboxylic acidfunctionalized variable organic moiety in the presence of a carbodiimidecatalyst can also be employed.

V. Properties of a Polymeric Sorbent of the Present Invention

The following sections provide additional detail of the properties of apolymeric sorbent of the present invention, as well as methods ofcharacterizing a polymeric sorbent that was prepared by a method of thepresent invention.

V.A. Structural Characterization of a Polymeric Sorbent Prepared by aMethod of the Present Invention

When preparing a polymeric sorbent of the present invention, it can bedesirable to confirm the incorporation of a particular functionality(e.g. an amide functionality) into the sorbent, as well as to determinethe overall composition and structure of the sorbent. Variousspectrophotometric and spectrometric techniques can be employed in thisregard. For example, FTIR and solid state ¹³C NMR spectroscopytechniques can be employed in the evaluation of a polymeric sorbent ofthe present invention include. Such techniques are known to those ofordinary skill in the art and, in the context of the present invention,can generally be employed as follows.

A functionalized polymer of the present invention (e.g., an amidefunctionalized polymer) can be characterized by its Fourier-transforminfrared (FTIR) spectrum and its solid state ³¹C NMR spectrum. Examplesof FTIR and solid state ¹³C NMR spectra acquired from a polymericsorbent are provided in FIG. 2 and FIG. 3A, respectively, for anacetamide functionalized polymeric sorbent.

Turning first to FIG. 2, several features of this FTIR spectrum arenotable. For example, the peak at about 3000 wavenumbers ischaracteristic of a methyl C-H stretching vibration and the peak at 3200cm⁻¹ is attributable to an N—H stretching vibration. The envelope atabout 1640 wavenumbers is characteristic of the amide carbonyl groupstretching vibration, while the bands in the 900-700 wavenumber regioncan be ascribed to di- and mono-substituted benzene rings. The peaksaround 1200 wavenumbers are characteristic of methylene rockingvibrations. Together, these structural features are indicative of thepresence of an acetamide functionalized poly(styrene divinylbenzene), anembodiment of a polymeric sorbent of the present invention.

Turning next to FIG. 3A, again several features of this solid state ¹³Cspectrum serve to characterize the functionalized styrene divinylbenzenepolymer. For example, the large peak around 40 ppm, together with thesmaller peaks at 29 and 15 ppm are attributable to exocyclic carbons onthe aromatic nucleus, while the peaks around 125-140 ppm belong toaromatic ring carbons. The peak around 24 ppm is indicative of themethyl carbon of the acetamide functionality. The downfield peaks ataround 150 and 170 ppm are characteristic of carbons situated near theamide nitrogen atom and carbonyl carbons. The peaks found around 70-90ppm and 180-200 ppm region arise from spinning sidebands, as was provenby their change in position/intensity when the spinner speed is changed.Considered cumulatively, these structural features are indicative of thepresent of an acetamide functionalized poly(styrene divinylbenzene),which is an embodiment of a polymeric sorbent of the present invention.

V.B. Solvation Properties of a Polymeric Sorbent of the PresentInvention

Several polymeric sorbents documented in the literature are reported toexhibit a high degree of surface hydration (see, e.g., Leon-Gonzalez &Perez-Arribas, (2000) J. Chromatogr. A 902: 3-16; Huck & Bonn, (2000) J.Chromatogr. A 885: 51-72; U.S. Pat. No. 5,618,438). Some of thesepolymers comprise a poly(styrene divinylbenzene) backbone and can carryfunctionalities such as a sulfonic or carboxylic acid moiety, a nitrogroup, a methyl or phenyl ketone, a hydroxy-methyl group, a quarterneryammonium, or a carboxy-substituted porphyrin moiety. However, theseprior art sorbents are not adapted to remain solvated for long periodsof time, for example longer than about one hour, while retaining theirseparative properties.

In one aspect of the present invention, an amide functionalityassociated with a polymeric backbone (e.g. a poly(styrenedivinylbenzene) backbone), which can be an acetamide group, enhances thesolvation (e.g. water wettability) of the surface of a polymeric sorbentof the present invention. The enhancement in the salvation is ofsignificance because plasma samples are typically loaded onto SPEsorbents in aqueous solutions and an efficient extraction of a drugcontained in the plasma sample will not take place when the sorbent isnot wetted (i.e. solvated) or is only partially wetted. Furthermore,after sample loading, the sorbent is typically washed with aqueoussolvents, and thorough washing of the drug adsorbed on the sorbentcannot be done without proper wetting of the surface. In addition,configurational changes in the structure/morphology of the sorbent canoccur, if the sorbent cannot remain wetted or is not completelywettable.

Even after subjecting a water-wetted polymeric sorbent of the presentinvention to a vacuum for one hour, the hydration level of thefunctionalized polymer is not significantly affected. Indeed, aftersolvation, a polymeric sorbent of the present invention can remainsolvated for at least about an hour in the absence of solvent. Thisproperty is exhibited in FIG. 7 for a seven component pharmaceuticalprobe mixture. FIG. 7 demonstrates that the recovery yield of the drugsis significantly enhanced (5 to 30%, maximum observed for the drugmianserin) when the sorbent remains wetted for longer periods afterconditioning with water and before the introduction of the sample ontothe sorbent. This is due to the fact that a more efficient interactionof the analyte with the sorbent surface takes place when the sorbentreaches equilibrium wettability condition. If the sorbent dries upduring standing, drug retention will be significantly altered andwashing and elution also become inefficient.

VI. Method of Isolating an Analyte

A polymeric sorbent that is employed in the method comprises a polymericsorbent of the present invention. As noted hereinabove, the polymericbackbone can comprise any polymer, with the caveat that the polymer beadapted to form at least one of a dipolar interaction and a hydrophobicinteraction. A representative, but non-limiting list of polymericbackbone constituents includes, but is not limited to, poly(styrenedivinylbenzene), poly(styrene divinylbenzene) functionalized with polargroups such as amide carrying variable organic moieties (e.g. furan ornitrophenyl or ester or ether which can enhance □—□ interactions); ahydroxyphenyl or amidophenyl moiety that can enhance the acidity of thesurface for reaction with basic samples; and a basic (e.g. nitrogencontaining heterocyclic) moiety that can interact with an acidic sample.A polymeric backbone (and subsequently a polymeric sorbent of thepresent invention) can, but need not, comprises particles having acharacteristic dimension (e.g. a diameter) of between about 20 and about120 microns. Furthermore, when a polymeric backbone comprises particles,it is the particles can comprise a pore size of, for example, betweenabout 50 to about 150 angstroms, or between about 50 to about 70angstroms.

Additionally, a polymeric sorbent of the present invention comprises, inone embodiment, an amide functionality adapted to undergo protonaccepting and proton donating interactions and is associated with thebackbone. In other embodiments, the amide functionality can comprise ahydrogen atom and a variable organic group, which itself can comprise amethyl group, making the amide functionality an acetamide group.

In another aspect of the present invention, a method of isolating ananalyte from a sample is disclosed. The sample can be derived from anysource, although the polymeric sorbent and methods of the presentinvention are particularly suited for isolating an analyte frombiological, environmental and pharmaceutical samples. For example, asample can comprise biological matrix (e.g., whole blood or plasma orsaliva or urine) comprising an analyte of interest (such as a drug).Alternatively, a sample can comprise an environmental sample, such asdrinking water or water known or suspected of being polluted. In anotherexample, the analyte of a pharmaceutical sample can comprise atherapeutically-active agent carried by a pharmaceutically-acceptableexcipient.

In this embodiment, the method can comprise four general steps:conditioning the sorbent with solvents that enhance surfacecharacteristics, loading the sample contained in an aqueous medium,washing the sample loaded sorbent with an appropriate binary (aqueousorganic) solvent and elution with a strong organic solvent.

In this embodiment, the method comprises conditioning the sorbent bywashing the sorbent with an organic conditioning solvent followed bywater. The sorbent can be associated with a support, such as a column,in which case, the step of conditioning can comprise passing an organicsolvent over the column, followed by passing an aqueous solvent over thecolumn. The initial conditioning step can be carried out by treating thesorbent with methanol and then with water (for example about 1 mL each).The methanol swells the sorbent and enhances the effective surface area.The water treatment removes excess methanol and also hydrates thesurface. The conditioned surface can then be subjected to vacuum toremove excess solvents; the sorbent remains completely hydrated afterthis treatment.

Continuing with the embodiment, a sample comprising an analyte can thenbe contacted with a polymeric sorbent comprising a polymeric backboneadapted to form at least one of a dipolar interaction and a hydrophobicinteraction and an amide functionality associated with the backbone andadapted to undergo proton accepting and proton donating interactions toform a sorbent-sample complex. This step, sometimes referred to assample loading, allows an association of sample, which can comprise ananalyte, with a sorbent. The larger the number/nature of interactions ofa sample with a sorbent surface, the greater its retention will be.Thus, a sorbent that facilitates a larger number and variety ofinteractions with a sample will strongly retain the sample.

When a sample comprises a plasma sample, the sample can be introduced asdiluted aqueous solutions (at least 1:1 dilution). This practice can bedesirable because of the high viscosity of as-obtained plasma samplesfrom animals or humans, which prevents free flow unless diluted toreduce viscosity. The use of organic solvents in this step is preferablyavoided, since these solvents can precipitate proteins from the plasmasolution and the precipitated proteins can foul the sorbent surface. Itcan also be desirable that a sample is contacted with a polymericsorbent under conditions conducive to the formation of an associationbetween an analyte and the polymeric sorbent. At the same time, theseconditions are preferably unfavorable for retaining unwanted proteinsand other impurities on the sorbent surface. Such conditions can includeconducting the contacting at about room temperature and neutral pH.

In a one embodiment of the present invention, a sample is loaded in a1:1 aqueous solution and an analyte (e.g. a drug) can be present in onenanogram to 10 microgram per milliliter levels. A sample volume of about100 to about 1000 microliters can be loaded, although volumes of about400 to about 500 microliters are preferred.

The sorbent-sample complex can then be washed with water, followed by anorganic wash solvent. This step can impact the cleanliness of the finaleluted sample. In fact, this is one aspect in which the material of thepresent invention performs at a superior level compared with known SPEsorbents, including polymeric second generation sorbents. In oneembodiment, the sample loaded sorbent is washed with water and then withabout 10 to about 30% acetonitrile in water (any volume can be employed,although volumes from about 200 to about 1000 microliters arepreferred). The water wash removes salts and other water-soluble matrixconstituents that might be present in a sample, in addition toproteinaceous matter. The binary aqueous-organic wash can also removeorganic impurities including water-insoluble matrix components that canadhere to the sorbent surface. It can be desirable to configure thiswash so as not to disrupt the binding of an analyte to the sorbentsurface. When many known silica based and polymeric sorbents areemployed in a separation, such a binary wash can remove many polaranalytes from the sorbent.

Next, an analyte is eluted from the sorbent-sample complex with aneluting solvent. The elution can be performed by passing a volume of aneluting solvent over a sorbent that has been contacted with a sample andwith which a sample is associated. Representative eluting solventsinclude binary solvents comprising an aqueous component and an organiccomponent. Preferably, the organic component comprises at least about80-90% of the solvent. Representative organic components include, butare not limited to, acetonitrile and methanol. A trailing ion, such astrifluoroacetic acid, can also be employed as a component of an elutionsolvent and serves to disrupt the polar interactions of polar drugs withthe sorbent effectively. In one embodiment of the present invention, a60:30:10 methanol/acetonitrile/0.1% trifluoroacetic acid is found toafford 90% to almost quantitative recoveries of drugs of a wide range ofpolarities (see FIG. 8). Eluting solvent volumes of about 400microliters to about 1000 microliters can be employed, and volumes about400 to about 500 microlitres are preferred in some situations.

The eluent can be collected and the identity of an adherent analyteascertained, for example by mass spectrometry, liquid chromatography,gas chromatograpy or a combination of these and other techniques knownto those of ordinary skill in the art. When an analyte of interest (e.g.a drug) is present in picogram levels in plasma, the eluting solvent canbe evaporated and the residual analyte redissolved (i.e. reconstituted)in about 40 to about 100 microliters of the mobile phase used for LC orLC/MS.

An advantage of the polymeric sorbents and associated methods of thepresent invention is the ability to pass eluent directly to aninstrument(s) for analyte identification. This is not possible with manyprior art sorbents, due, in part, to ion suppression effects of priorart sorbents and the inability of these sorbents to retain moderatelypolar to highly polar analytes. These deficiencies can lead to unwantedcomponents in an eluent, which can significantly complicate analyteidentification operations, and poor MS spectra. For example, a sorbentof the present invention can form a component of a system comprising thesorbent and a LC/MS/MS system. Samples can be loaded onto the sorbent,analytes eluted and the eluent stream fed directly into an LC/MS/MSsystem, HPLC system or any of a range of analytical instruments.

In yet another embodiment of a method of the present invention, thesorbent can be associated with a support or supporting format. A list ofrepresentative supports and supporting formats includes syringe barrelcartridges, polymeric fiber membranes, glass fiber membranes andmultiwelled plates, although disks and other supports can also beemployed. The sorbent can be disposed on the surface of a supportingformat, for example on the surface of a multiwelled plate, or thesorbent can be embedded in a supporting format, for example in apolymeric or glass fiber membrane. Thus, by “association” it isgenerally meant that a sorbent can be in contact with a support orsupporting format.

VII. Comparative Examples

In one comparative example, the ability of a polymeric sorbent of thepresent invention, namely an acetamide functionalized poly(styrenedivinylbenzene), to isolate the same combination of eight drugs asmentioned in FIG. 8 from a biological matrix, namely plasma derived froman animal, was investigated. This efficiency was compared with theefficiency of a commercially available prior art polymeric sorbent toperform the same task, namely a divinylbenzene-N-vinylpyrrolidone resin(commercialized as OASIS® and available from Waters Corporation,Milford, Mass.). FIGS. 9 and 10 summarize the results of thiscomparative example. These figures show a comparison in the mass ranges500 to 2200 and 400 to 800, respectively.

Continuing with the comparative example, FIGS. 9 and 10 indicate thatthe OASIS® resin retains significant proportions of matrix constituentsfrom plasma, which show up as a group of peaks in the mass spectra bothin the lower and higher mass regions. For the OASIS® sorbent, the SPEprocedure recommended by the manufacturers was followed, while for thepolymer of the present invention, the method described in the previoussection was employed. The X-axis in both figures represents mass and theY-axis denotes intensity of each peak as “counts”.

In another comparative example, the ion suppression of the samepolymeric sorbent of the present invention, namely an acetamidefunctionalized poly(styrene divinylbenzene), was compared with the ionsuppression of a commercially available polymeric sorbent, namely theOASIS® sorbent. FIG. 11 presents the results of this comparison.Summarily, FIG. 11 indicates that the polymeric sorbent of the presentinvention exhibits very low ion suppression under LC/MS/MS analysisemploying the ES ionization mode of a mass spectrometric detector.

In one aspect, FIG. 11 indicates that when a polymeric sorbent of thepresent invention is employed, the minimum part of the curve is muchsmaller than that achieved when the OASIS® sorbent is employed, and thatthe curve returns from the minimum point to the original level (startinglevel) very quickly (in less than 1 min). By way of comparison, when theOASIS® sorbent is employed, it takes more than 10 min (X-axis indicatestime in minutes) to do so. This observation indicates that if there areany peaks from analytes (drugs) appearing in this region, they fallunder the influence of matrix constituents exhibiting this suppressioneffect and their intensities are affected.

The data of this comparative example was generated by pumping a constantconcentration of a drug (i.e. mianserin) through the LC/MS system at aconstant rate and infusing a plasma extract that has been purified byrunning through the appropriate SPE sorbent into the mobile phase afterit had passed through the LC column, and before it enters the massspectrometer. Since the mobile phase contains a constant level ofmianserin, ion suppression due to the infusion (injection) of the plasmaextract would result only if there are matrix constituents present inthe plasma extract purified by passing through the sorbent.

Laboratory Examples

The following Laboratory Examples have been included to illustratepreferred modes of the invention. Certain aspects of the followingLaboratory Examples are described in terms of techniques and proceduresfound or contemplated by the present inventors to work well in thepractice of the invention. These Laboratory Examples are exemplifiedthrough the use of standard laboratory practices of the inventors. Inlight of the present disclosure and the general level of skill in theart, those of skill will appreciate that the following LaboratoryExamples are intended to be exemplary only and that numerous changes,modifications and alterations can be employed without departing from thespirit and scope of the present invention.

Laboratory Example 1 Preparation of an Acetamide FunctionalizedPoly(styrene divinylbenzene,

1.A. Nitration

Poly(styrene divinylbenzene) beads (1 mole) was suspended inconcentrated nitric acid (30 molar equivalent) and the mixture wasmechanically stirred at a low rpm (100-200 rpm) to prevent breakage ofthe beads. While cooling the mixture in cold water, concentratedsulfuric acid (18 molar equivalent) was added dropwise over a period of1 to 1.5 hours, continuing the stirring at the same time. The mixturewas further stirred at room temperature for three more hours. Thereaction mixture was poured into 10-12L of deionized water and afterstirring with a polytetrafluoroethylene (commercially available asTEFLON® from DuPont, Wilmington, Del.) tipped rod, the suspension wasallowed to stand for 16 hours. The nitrated polymer was recovered byfiltering through a sintered glass funnel under vacuum and washed with2.0 M sodium hydroxide initially, followed by 1.0 M sodium hydroxideseveral times and finally with deionized water until the filtrate was nolonger basic. The product was then rinsed with acetone and dried undervacuum at 70-80° C.

1.B. Reduction

The nitrated poly(styrene divinylbenzene) was suspended in acetic acid(2.5L) and while mechanically stirring at a low rpm, (100-200 rpm)treated with a solution of stannous chloride (1.25 kg) in 1:1hydrochloric acid (3L). The mixture was stirred at room temperature for60 hours. The polymer was recovered by filtration, washed first withdeionized water and then 1.0M sodium hydroxide several times till notrace of tin was found in the filtrate. Then the polymer was washed withwater until the filtrate is neutral, and then washed with acetone. Theproduct was dried under vacuum at 70-80° C.

1.C. Acetylation

The aminated poly(styrene divinylbenzene) was suspended in a base(triethylamine or pyridine, excess) and with slow mechanical stirring,treated dropwise with acetic anhydride (1. 1 mole equivalent to thepolymer). The stirring was continued for 3.5 hours. The functionalizedpolymer was recovered by filtration and washed several times with 0.1Mhydrochloric acid and then with deionized water until the filtrate wasneutral. The polymer was washed with methanol several times and thenwashed with acetone two to three times. Finally, the polymer was driedunder vacuum at 70-80° C.

Laboratory Example 2 Solid Phase Extraction Of Canine Plasma SampleSpiked With Pharmaceutical Probes

The amide functionalized poly(styrene divinylbenzene) sorbent (10 mg)prepared in Laboratory Example 1 was slurry packed into syringe barrelcartridges or a 96 well plate (Ansys/Varian Inc., Palo Alto, Calif.)with water as a slurry solvent. The sorbent was conditioned with 1 mL ofmethanol, followed by 1 mL of deionized water. A plasma sample spikedwith pharmaceutical probes (1:1 diluted, 200 microliters) was thenloaded with the application of a gentle vacuum. The cartridge or welledplate was then washed with 1 mL of 10-20% acetonitrile in water under agentle vacuum. The drugs (analytes) were then eluted with 400-700microlitres of methanol-water (95:5) or methanol/acetonitrile/water(60:40: 10) containing 0.1% of formic or acetic or trifluoroacetic acid.Optionally, the extract could have been concentrated under vacuum andreconstituted in 200 microliters of methanol or acetonitrile, or amixture of these two solvents, with or without 0.1% of formic or aceticacid. The reconstituted extract was analyzed either by HPLC with UVdetection or with LC/MS/MS on a Varian 1200L or PE Sciex API III massspectrometer detector (Varian Inc., Palo Alto, Calif.).

The recoveries of eight analytes with a wide spectrum of polarities froma canine plasma sample spiked with these pharmaceuticals by solid phaseextraction are shown in FIG. 8. The drugs studied comprise of a widespectrum of polarities, ranging from a log P value of 0.0 to 0.5 foratenolol and ranitidine, to 1.5 for pseudoephedrine, 2.5 for quinidineand 3.0 to 5.0 for brompheneramine, mianserin and fluoxetine.Haloperidol (used as an internal standard) also falls into thehydrophobic drug category. The figure clearly shows that recoveries inthe range of 60-70% can be achieved with as small an eluting solventvolume as 400 microliters. Utilizing 1 mL of eluting solvent, therecoveries of all the drugs jumps to about 92-94% for the polar drugsand 81 to 100% for the hydrophobic drugs. In contrast, the recoveries ofpolar drugs is in the 12 to 19% range when the OASIS® sorbent isemployed. These SPE experiments unequivocally demonstrate the universalnature of the polymer of the present invention with equalrecoveries/retention for polar, moderately polar and hydrophobic drugs.

Laboratory Example 3 Drying a Sorbent Followed by SPE

Laboratory Example 3 was conducted in the same manner as described forLaboratory Example 2, with the exception that the conditioned cartridgewas dried under gentle vacuum for one hour prior to sample loading. Theresults of performing an SPE protocol after drying a sorbent of thepresent invention are presented in FIG. 7.

References

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It will be understood that various details of the invention may bechanged without departing from the scope of the invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation—the invention being defined by theclaims.

1. A polymeric sorbent comprising: (a) a polymeric backbone adapted tofacilitate one or more interactions selected from the group consistingof a dipolar interaction and a hydrophobic interaction; and (b) an amidefunctionality associated with the polymeric backbone via a covalent bondand adapted to undergo one or more interactions selected from the groupconsisting of proton accepting, proton donating and dipolarinteractions.
 2. A polymeric sorbent comprising: (a) a polymericbackbone adapted to facilitate one or more interactions selected fromthe group consisting of a dipolar interaction and a hydrophobicinteraction; and (b) an amide functionality associated with thepolymeric backbone and adapted to undergo one or more interactionsselected from the group consisting of proton accepting, proton donatingand dipolar interactions, wherein the nitrogen atom of the amidefunctionality is associated with the polymeric backbone, hydrogen atomor an alkyl, aryl or heterocyclic moiety and a variable organic groupcomprising a carbonyl and an alkyl, cycloalkyl, aryl, or heterocyclicgroup.
 3. The polymeric sorbent of claim 2, wherein the polymericbackbone is selected from the group consisting of poly(styrenedivinylbenzene), copolymers of styrene, copolymers of divinylbenzene,functionalized styrenes, functionalized heterocycles and combinationsthereof.
 4. The polymeric sorbent of claim 2, wherein the amidefunctionality is selected from the group consisting of acetamide,N-alkylamides, N-aryl amides, and N-heteryl amides.
 5. The polymericsorbent of claim 2, wherein the polymeric sorbent comprises betweenabout 3.5% and about 5.0% nitrogen by mass percent.
 6. The polymericsorbent of claim 2, wherein the polymeric sorbent comprises particleshaving a characteristic dimension of between about 20 and about 120microns.
 7. The polymeric sorbent of claim 2, wherein the polymericsorbent is adapted to remain solvated for longer than about one hourafter contact with a solvent.
 8. The polymeric sorbent of claim 2,wherein the polymeric sorbent is adapted to adsorb strongly polar,moderately polar or nonpolar molecules.
 9. The polymeric sorbent ofclaim 2, wherein the polymeric sorbent is associated with a support. 10.The polymeric sorbent of claim 9, wherein the support is selected fromgroup consisting of a cartridge, a polymeric fiber membrane, a glassfiber membrane and a multi-well plate.
 11. The polymeric sorbent ofclaim 1, wherein the polymeric backbone is selected from the groupconsisting of poly(styrene divinylbenzene), copolymers of styrene,copolymers of divinylbenzene, functionalized styrenes, functionalizedheterocycles and combinations thereof.
 12. The polymeric sorbent ofclaim 1, wherein the amide functionality is selected from the groupconsisting of acetamide, N-alkylamides, N-aryl amides and N-heterylamides.
 13. The polymeric sorbent of claim 1, wherein the polymericsorbent comprises between about 3.5% and about 5.0% nitrogen by masspercent.
 14. The polymeric sorbent of claim 1, wherein the polymericsorbent comprises particles having a characteristic dimension of betweenabout 20 and about 120 microns.
 15. The polymeric sorbent of claim 1,wherein the polymeric sorbent is adapted to remain solvated for longerthan about one hour after contact with a solvent.
 16. The polymericsorbent of claim 1, wherein the polymeric sorbent is adapted to adsorbstrongly polar, moderately polar or nonpolar molecules.
 17. Thepolymeric sorbent of claim 1, wherein the polymeric sorbent isassociated with a support.
 18. The polymeric sorbent of claim 17,wherein the support is selected from the group consisting of acartridge, a polymeric fiber membrane, a glass fiber membrane and amulti-well plate.
 19. A method of preparing a polymeric sorbentfunctionalized with an amide functionality, the method comprising: (a)nitrating a polymeric backbone to form a nitrated polymeric backbone;(b) reducing the nitrated polymeric backbone to form an aminatedpolymeric backbone; and (c) contacting the aminated polymeric backbonewith one of an acid, an acid chloride or an acid anhydride.
 20. Themethod of claim 19, wherein the polymeric backbone is adapted to undergodipolar and hydrophobic interactions with an analyte.
 21. The method ofclaim 19, wherein the polymeric backbone is selected from the groupconsisting of poly(styrene divinylbenzene), copolymers of styrene,copolymers of divinylbenzene, functionalized styrenes, functionalizedheterocycles and combinations thereof.
 22. The method of claim 19,wherein the nitrating comprises: (a) suspending the polymeric backbonein a first solution comprising nitric acid; and (b) adding a secondsolution comprising a reagent adapted to generate a nitronium ion to thefirst solution.
 23. The method of claim 19, wherein the step of reducingcomprises: (a) suspending the nitrated polymeric backbone in a firstsolution comprising a first acid; and (b) contacting the nitratedpolymeric backbone with a second solution comprising a metal catalystand a second acid.
 24. The method of claim 23, wherein the first acid isan organic acid.
 25. The method of claim 24, wherein the second acid isselected from the group consisting of hydrochloric acid, an organic acidand combinations thereof.
 26. The method of claim 23, wherein the metalcatalyst is selected from the group consisting of stannous chloride,zinc metal, an organo-metallic hydride, and hydrogen in the presence ofa metal.
 27. The method of claim 19, wherein the contacting comprises:(a) suspending the reduced polymeric backbone in a first solutioncomprising a base to form a basic reaction solution; and (b) adding oneof an acid, an acid chloride and an anhydride to the basic reactionsolution.
 28. The method of claim 27, wherein the base is selected fromthe group consisting of triethylamine, pyridine, alkyl pyridines,quinoline, alkylquinolines, trialkylamines, imidazole and triazole. 29.The method of claim 27, wherein the acid chloride is selected from thegroup consisting of acetyl chloride, alkanoyl chlorides, aryl chloridesand heteryl chlorides.
 30. The method of claim 27, wherein the anhydrideis selected from the group consisting of acetic anhydride, anhydrides ofhigher aliphatic acids, anhydrides of aromatic acids, anhydrides ofheterocyclic acids and mixed anhydrides.
 31. The method of claim 27,wherein the acid is selected from the group consisting of aliphaticacids, aromatic acids, and heterocyclic carboxylic acids.
 32. The methodof claim 19, wherein the amide functionality is adapted to undergoproton donating and proton accepting interactions.
 33. The method ofclaim 19, wherein the polymeric sorbent comprises between about 3.5% andabout 5.0% nitrogen by mass percent.
 34. The method of claim 19, whereinthe polymeric sorbent comprises particles having a characteristicdimension of between about 20 and about 120 microns.
 35. The method ofclaim 19, wherein the polymeric sorbent formed by the method remainssolvated, after contact with one of water and an organic solvent, forlonger than about one hour.
 36. The method of claim 19, wherein thepolymeric sorbent is adapted to adsorb strongly polar, moderately polarand nonpolar molecules.
 37. The method of claim 19, further comprising:(a) recovering the polymeric sorbent by filtration; (b) washing thepolymeric sorbent one or more times with a solution comprising an acid;(c) washing the polymeric sorbent one or more times with an aqueoussolution; and (d) washing the polymeric sorbent one or more times withan organic solvent.
 38. A polymeric sorbent produced by the method ofclaim 19.